Haemophilus ducreyi is the etiologic agent of chancroid. The current understanding of the pathogenesis of chancroid is limited. Recently, the genes for the hemolytic cytotocin of H. ducreyi have been cloned. Further, the hemolytic cytotoxin has been shown to be responsible for the cytopathic effect observed on human foreskin fibroblasts. The aim of this study is to address the factors responsible for the regulation of the hemolysin. In our preliminary studies, we have demonstrated that hemolysin is elaborated during log phase growth and is dependent on the concentration of heme in the medium. To more conveniently characterize the regulation of hemolysin expression, a H. ducreyi mutant will be constructed that contains a reporter gene, lacZ, under the control of the hemolysin promoter. Such a construct will allow a more efficient measurement of the transcription of the hemolysin gene cluster and will aid in defining what environmental factors regulate hemolytic activity. The hemolysin will be further characterized at the transcriptional level. mRNA will be measured to determined if the hemolysin genes are transcribed as an operon or as two separate transcripts. Furthermore, the start site of the single or both mRNA fragments will be mapped. Since virulence in chancroid is undoubtly a multifactorial process, an understanding of eh regulation of hemoloysin expression will lead to the identification of other co-regulated virulence determinants.